The present invention relates to novel radioligands and to methods for detecting specific activity of compounds. More particularly, the present specification discloses assays for detecting the binding of compounds to sites that regulate potassium channels.
Potassium channels play important roles in regulating cell membrane excitability. In particular, ATP-sensitive potassium channels that are inhibited by intracellular ATP link cellular metabolism with membrane excitability in various cell types including cardiac, smooth muscle, neurons and secretory cells. In these cell types, KATP channels modulate physiological processes including insulin secretion from pancreas, leptin release from hypothalamic neurons, synaptic transmission and excitability of cardiac, vascular and nonvascular smooth muscles. Openers of various KATP channels can alter the cell""s resting membrane potential and in turn cellular excitability to regulate these diverse processes. A number of diseases or conditions can be treated with therapeutic agents that open potassium channels. Such diseases or conditions include asthma, epilepsy, hypertension, sexual dysfunction, pain, migraine, urinary incontinence, stroke and neurodegeneration.
[3H]P1075 is a known radioligand for the ATP-sensitive potassium channel described by Brya et al., 1992. [3H]Bay X 9228 is another known radioligand for the ATP-senstive potassium channel. U.S. Pat. No. 5,328,830 discloses the utilization of tritiated (+)-N-(2-ethoxyphenyl)-Nxe2x80x2-(1,2,2-trimethyl propyl)-2-nitroethene-1,1-diamine ([3H]CMPD), for assaying compounds whose activity is specific for ATP-sensitive potassium channels. However, the utility of these ligands are limited. These assays generally require intact cell or tissue preparations with large inherent variability due to poor specific activity and poor binding affinities Loffler-Walz, C., Quast, U. (1998) Br. J. Pharmacol., 123, 1395-1402.
Accordingly, a novel radioligand with improved potency and higher specific activity would be useful for characterization of KATP channels in various tissues in addition to further investigation of the mechanism of action of novel potassium channel modulators with native and recombinant KATP complex. The present invention relates to novel 1,4-dihydropyridine radioligands that have higher affinities and higher specific activity in interacting with the ATP-sensitive potassium channel. Specifically, the present invention discloses assays using novel 1,4-dihydropyridine radioligands for screening and identification of compounds that interact with ATP-sensitive potassium channels.
In one embodiment, the present invention discloses compounds of formula I: 
or salts thereof wherein
n is an integer of 0-1;
m is an integer of 1-2;
provided that when n is 1, then m is 1;
A is selected from C(O) and S(O)2;
D is selected from O, S and CR2R3;
Z is selected from O and S;
R1 is selected from alkyl, cyano, haloalkoxy, haloalkyl, halogen and nitro; and
R2, R3, R4, R5, R6 and R7 are each independently selected from hydrogen and alkyl;
provided that when A is S(O)2; then D is CR2R3; and
further provided that when D is S; then n is 1 and A is C(O).
The compounds of the present invention are novel radioligands that bind to the ATP sensitive potassium channels. The present invention also relates to a binding assay that can be used to evaluate compounds that bind to a site that regulates the function of the K+ channel complex. These compounds are useful for high throughput screening of compound libraries to identify novel ligands interacting with the ATP-sensitive potassium channels. Such radioligands could also be of utility in: (i) characterization of recombinant ATP-sensitive channel subunits; (ii) use as a tool to identify novel KATP channel subunits (iii) study distribution of KATP channels in situ in various tissues in physiological and disease states and (iv) in vivo imaging of ATP-sensitive potassium channels, including photoemission computed tomography.
In one embodiment, the present invention discloses compounds of formula I: 
or salts thereof wherein
n is an integer of 0-1;
m is an integer of 1-2;
provided that when n is 1, then m is 1;
A is selected from C(O) and S(O)2;
D is selected from O, S and CR2R3;
Z is selected from O and S;
R1 is selected from alkyl, cyano, haloalkoxy, haloalkyl, halogen and nitro; and
R2, R3, R4, R5, R6 and R7 are each independently selected from hydrogen and alkyl;
provided that when A is S(O)2; then D is CR2R3; and
further provided that when D is S; then n is 1 and A is C(O).
In another embodiment, compounds of the present invention have formula (I) wherein R1 is selected from alkyl, haloalkyl and halogen; R4, R5, R6 and R7 are each hydrogen; and m, n, A, D and Z are as defined in formula (I).
In another embodiment, compounds of the present invention have formula (I) wherein n is 0; A is S(O)2; D is CR2R3; and m, Z, R1, R2, R3, R4, R5, R6 and R7 are as defined in formula (I).
In another embodiment, compounds of the present invention have formula (I) wherein m is 1; n is 0; A is S(O)2; D is CR2R3; Z is O; R2, R3, R4, R5, R6 and R7 are each hydrogen; and R1 is as defined in formula (I).
In another embodiment, compounds of the present invention have formula (I) wherein n is 0; A is C(O); D is CR2R3; and m, Z, R1, R2, R3, R4, R5, R6 and R7 are as defined in formula (I).
In another embodiment, compounds of the present invention have formula (I) wherein n is 0; A is C(O); D is O; and m, Z, R1, R2, R3, R4, R5, R6 and R7 are as defined in formula (I).
In another embodiment, compounds of the present invention have formula (I) wherein m is 1; n is 0; A is C(O); D is O; Z is O; R4, R5, R6 and R7 are each hydrogen; and R1 is as defined in formula (I).
In another embodiment, compounds of the present invention have formula (I) wherein n is 0; A is C(O); D is O; R4, R5 and R6 are each hydrogen; R7 is alkyl; m, Z and R1 are as defined in formula (I).
In another embodiment, compounds of the present invention have formula (I) wherein m is 1; n is 0; A is C(O); D is O; Z is O; R4, R5 and R6 are each hydrogen; R7 is alkyl; and R1 is as defined in formula (I).
In another embodiment, compounds of the present invention have formula (I) wherein m is 1; n is 1; A is C(O); D is O; and Z, R1, R4, R5, R6 and R7 are as defined in formula (I).
In another embodiment, compounds of the present invention have formula (I) wherein m is 1; n is 1; A is C(O); D is O; R4, R5, R6 and R7 are each hydrogen; and Z and R1 are as defined in formula (I).
In another embodiment, compounds of the present invention have formula (I) wherein m is 1; n is 1; A is C(O); D is O; Z is O; R4, R5, R6 and R7 are each hydrogen; and R1 is as defined in formula (I).
As used throughout this specification and the appended claims, the following terms have the following meanings:
The term xe2x80x9calkyl,xe2x80x9d as used herein, refers to a straight or branched chain hydrocarbon containing from 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl.
The term xe2x80x9ccyano,xe2x80x9d as used herein, refers to a xe2x80x94CN group.
The term xe2x80x9chaloxe2x80x9d or xe2x80x9chalogen,xe2x80x9d as used herein, refers to xe2x80x94Cl, xe2x80x94Br, xe2x80x94I or xe2x80x94F.
The term xe2x80x9chaloalkoxy,xe2x80x9d as used herein, refers to at least one halogen, as defined herein, appended to the parent molecular moiety through an alkoxy group, as defined herein. Representative examples of haloalkoxy include, but are not limited to, chloromethoxy, 2-fluoroethoxy, trifluoromethoxy, and pentafluoroethoxy.
The term xe2x80x9chaloalkyl,xe2x80x9d as used herein, refers to at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl.
The termer xe2x80x9cnitrogen protecting group,xe2x80x9d or xe2x80x9cN-protecting group,xe2x80x9d as used herein, refers to those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Nitrogen protecting groups comprise carbamates, amides, N-benzyl derivatives, and imine derivatives. Preferred nitrogen protecting groups are acetyl, benzoyl, benzyl, benzyloxycarbonyl (Cbz), formyl, phenylsulfonyl, pivaloyl, tert-butoxycarbonyl (Boc), tert-butylacetyl, trifluoroacetyl, and triphenylmethyl (trityl). Commonly used N-protecting groups are disclosed in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd edition, John Wiley and Sons, New York (1999).
The term xe2x80x9cnitro,xe2x80x9d as used herein, refers to a xe2x80x94NO2 group.
The radioligands of the present invention can be used in the form of salts derived from inorganic or organic acids. The salts can be prepared in situ during the final isolation and purification of the radioligands of the present invention or separately by reacting a free base function with a suitable organic acid. Examples of acids which can be employed to form salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid.
The compounds and processes of the present invention will be better understood by reference to the following Schemes and Examples, which are intended as an illustration of, and not a limitation upon the scope of the invention. Furthermore, all citations contained within this document are hereby fully incorporated by reference. In particular, U.S. Pat. No. 6,191,140 is fully incorporated by reference.
The compounds of this invention can be prepared by a variety of synthetic routes. Representative procedures are shown in Schemes 1-24. 
[125I]Dihydropyridines of general formula (3), wherein A, D, Z, R1, R4, R5, R6, R7, m and n are as defined in formula (I), can be prepared as described in Scheme 1. Compounds of general formula (1), wherein X is selected from Br or I, can be treated with a nitrogen protecting reagent such as di-tert-butyl dicarbonate in a solvent such as acetonitrile (MeCN) in the presence of 4-dimethylaminopyridine (DMAP) to provide N-protected dihydropyridines which can then be treated with a suitable tin reagent such as hexamethylditin in the presence of a palladium catalyst with heating in a solvent such as 1,4-dioxane to provide dihydropyridines of general formula (2). Dihydropyridines of general formula (2) can be treated with N-chlorosuccinimide (NCS) and an appropriate source of 125I such as Na125I in a mixture of acetic acid and methanol to provide N-protected [125I]dihydropyridines which can then be deprotected, for example with trifluoroacetic acid (TFA), to provide [125I]dihydropyridines of general formula (3). 
An alternative method of preparing [125I]dihydropyridines of general formula (3), wherein A, D, Z, R1, R4, R5, R6, R7, m and n are as defined in formula (I), can be used as described in Scheme 2. Compounds of general formula (1), wherein X is selected from Br or I, can be treated with a suitable tin reagent such as hexamethylditin in the presence of a palladium catalyst with heating in a solvent such as 1,4-dioxane to provide dihydropyridines of general formula (4). The conversion of compounds of general formula (1) to compounds of general formula (4) may require the presence of di-tert-butyl dicarbonate. Dihydropyridines of general formula (4) can be treated with N-chlorosuccinimide and an appropriate source of 125I such as Na125I in the presence of a mixture of acetic acid and methanol to provide [125I]dihydropyridines of general formula (3).
Schemes 3-24 illustrate intermediates and/or methods that can be used for generating dihydropyridines of general formula (1). Dihydropyridines of general formula (1) can be processed as described in Scheme 1 or Scheme 2 to generate [125I]dihydropyridines of general formula (3). 
Dihydropyridines of general formula (1), wherein A, D, Z, R1, R4, R5, R6, R7, m and n are as defined in formula (I), and X is Br or I, can be prepared as described in Scheme 3. Dicarbonyl compounds of general formula (10), aldehydes of general formula (11), and carbonyl compounds of general formula (12) can be combined in the presence of ammonia with heating in a solvent such as ethanol to provide dihydropyridines of general formula (1). An additional heating step, with an acid such as HCl, may be required to drive the reaction to completion.
Mixtures of isomers, that result from the synthetic methodology described in Scheme 3 as well as isomers generated in the Schemes that follow, can be separated by methods known to those skilled to the art. 
Dihydropyridines of general formula (15), wherein D, Z, R1, R4, R5, R6 and R7 are as defined in formula (I), wherein X is selected from Br or I, can be prepared as described in Scheme 4. One of the dicarbonyl components (10) or (14) can be treated with ammonia followed by addition of aldehydes of general formula (11) and the other dicarbonyl compound (10) or (14) with heating to provide dihydropyridines of general formula (15). An additional heating step, with an acid such as HCl, may be required to drive the reaction to completion. 
Dicarbonyl compounds of general formula (20), wherein R4, R5, and Z are as defined in formula (I), can be prepared as described in Scheme 5. Esters of general formula (17) can be alkylated with chloroacetone to provide ketoesters of general formula (19) wherein Z is sulfur. Ketoesters of general formula (19) can be cyclized in the presence of a base such as potassium tert-butoxide to provide dicarbonyl compounds of general formula (20). An alternative method of preparing ketoesters of general formula (19) can also be used. Acid chlorides of general formula (18), wherein Z is oxygen, prepared as described in (Terasawa, J. Org. Chem. (1977), 42, 1163-1169), can be treated with dimethyl zinc in the presence of a palladium catalyst in a solvent such as diethyl ether or tetrahydrofuran or toluene or a combination thereof to provide ketoesters of general formula (19).
An alternative method of preparing dicarbonyl compounds of general formula (20) can be used as described in Scheme 5. Alkynes of general formula (21) can be treated with methyl bromoacetate to provide compounds of general formula (22). A base such as sodium hydride may be necessary. Alkynes of general formula (22) can be treated with a catalyst such as mercuric acetate in the presence of a catalytic amount of sulfuric acid with heating in a solvent such as methanol followed by treatment with aqueous acid to provide methyl ketones of general formula (23). Methyl ketones of general formula (23) can be treated with a base such as potassium tert-butoxide to provide dicarbonyl compounds of general formula (20).
Alkynes of general formula (21), wherein Z is oxygen, can be purchased or prepared by reaction of a nucleophilic source of acetylene such a ethynylmagnesium bromide or lithium acetylide with an appropriate ketone or aldehyde.
Chiral alkynes of general formula (21), wherein Z is oxygen, can also be purchased or generated by known methods (Midland, M. Tetrahedron (1984), 40, 1371-1380; Smith, R. J.Med.Chem. (1988), 31, 1558-1566) and then processed to provide chiral dicarbonyl compounds of general formula (20).
Dicarbonyl compounds of general formula (20) may also be prepared using the procedures described in (Terasawa, T., Journal of Organic Chemistry 42 (1977)1163); Fehnel, J.Amer.Chem.Soc., (1955), 77, 4241-4242; Morgan, J.Amer.Chem.Soc. (1957), 79, 422; and Er, Helv.Chim.Acta, (1992), 75, 2265-2269). 
Dicarbonyl compounds of general formula (26), wherein R6 and R7 are defined as in formula (I) and D is selected from oxygen and sulfur, can be prepared as described in Scheme 6. Compounds of general formulas (25), (27) and (28) can be processed as described in Scheme 5 to provide dicarbonyl compounds of general formula (26). 
Dicarbonyl pyrans of general formula (35), wherein R4 and R5 are as defined in formula (I), can be prepared as described in Scheme 7. (Trimethylsilyl)acetylene can be deprotonated with a base such as n-butyllithium, methyllithium or ethyl magnesium bromide in a sovent such as diethyl ether or tetrahydrofuran and then treated with an aldehyde of general formula (30) to provide propargyl alcohols of general formula (31). Propargyl alcohols of general formula (31) can be treated with a base such as sodium hydride and then treated with methyl bromoacetate in a solvent such as tetrahydrofuran to provide alkynes of general formula (32). Alkynes of general formula (32) can be treated with a base such as lithium diisopropylamide or lithium bis(trimethylsilyl)amide and then treated with an alkylating agent such as an alkyl halide, alkyl sulfonate or the like in a solvent such as tetrahydrofuran to provide alkynes of general formula (33). Alkynes of general formula (33) can be treated with a source of mercury(II) such as mercury(II) acetate in the presence of acid in a solvent such as methanol followed by treatment with aqueous acid to provide methyl ketones of general formula (34). Methyl ketones of general formula (34) can be treated with a base such as potassium tert-butoxide to provide dicarbonyl pyrans of general formula (35).
Propargyl alcohols of general formula (31) can be separated into single enantiomers as described in (Burgess, J. Amer. Chem. Soc. (1990), 112, 7434-7435; Burgess, J. Amer. Chem. Soc. (1991), 113, 6129-6139; Takano, Synthesis (1993), 12, 1253-1256; and Allevi, Tetrahedron Assymmetry (1997), 8, 93-100). Single enantiomers of general formula (31) can be processed as described in Scheme 7 to provide enantiomerically pure dicarbonyl pyrans of general formula (35). 
Dicarbonyl thiopyrans of general formula (43), wherein R4 and R5 are as defined in formula (I), can be prepared as described in Scheme 8. Propargyl alcohols of general formula (31) can be converted to the corresponding chlorides using chlorinating agents well known to those of skill in the art such as phosphorous oxychloride and then treated with a source of sulfur such as sodium hydrogen sulfide to provide thiols of general formula (42) as described in (Komasov, Bull. Acad. Sci. USSR Div. Chem. Sci. (Engl. Trans.) (1963), 81-85). Thiols of general formula (42) can be processed as described in Scheme 7 to provide dicarbonyl thiopyrans of general formula (43).
Alternatively, dicarbonyl compounds of general formula (43) may be prepared using procedures as described in (Bergel, Nature(London) (1945), 155, 481). 
Dihydropyridines of general formula (46), wherein D, Z, R1, R4, R5, R6 and R7 are as defined in formula (I), wherein X is selected from Br or I, can be prepared as described in Scheme 9. One of the dicarbonyl components (10) or (45) can be treated with ammonia followed by addition of aldehydes of general formula (11) followed by treatment with the other dicarbonyl compound (10) or (45) with heating in a solvent such as ethanol to provide dihydropyridines of general formula (46). An additional heating step, with an acid such as HCl, may be required to drive the reaction to completion. Dicarbonyl compounds of general formula (45) can be prepared as described in (d""Angelo, Tett. Lett. (1991), 32, 3063-3066; Nakagawa, Heterocycles (1979), 13, 477-495; Sato, Masayuki, Tetrahedron: Asymmetry (1994), 5, 1665-1668; Sato, Masayuki, Tetrahedron Lett. (1990), 31, 7463-7466; Arnett, Edward M., J.Amer.Chem.Soc. (1987), 109, 809-812; Tabuchi, Hiroyasu, J.Chem.Soc.Perkin Trans.1 (1994), 125-134; Tabuchi, Hiroyasu, J.Org.Chem. (1994), 59, 4749-4759; Hofer, Roger, Helv.Chim.Acta (1985), 68, 969-974). 
Dihydropyridines of general formula (49), wherein Z, R1, R4, R5, R6, R7 and m are as defined in formula (I) and X is selected from Br or I, can be prepared as described in Scheme 10. One of the dicarbonyl components (10) or (48) can be treated with ammonia followed by addition of aldehydes of general formula (11) and the other dicarbonyl compound (10) or (48) with heating in a solvent such as ethanol to provide dihydropyridines of general formula (49). An additional heating step, with an acid such as HCl, may be required to drive the reaction to completion. Dicarbonyl compounds of general formula (48) can be purchased commercially or prepared using procedures similar to those described in (Stetter, Chem.Ber. (1958), 91, 374; Schinzer, Liebigs Ann.Chem.(1992), 2, 139-144; Knoevenagel, Chem.Ber.(1902), 35, 2182; Champagne, Can.J.Chem., (1964), 42, 212-222; Gilling, J.Chem.Soc. (1913), 103, 2033; Crossley, J.Chem.Soc.(1915), 107, 605; Kvita, Collect.Czech.Chem.Commun.(1957) 22, 1064; Hinkel, J.Chem.Soc.(1931), 814; Fadda, J.Indian Chem.Soc. (1990), 67, 915-917; Craig, J.Org.Chem.(1967), 32, 3743-3749; Deno, J.Amer.Chem.Soc. (1968), 90, 4085-4088; Matoba, Chem.Pharm.Bull.(1983), 2955-2956; Edafiogho, J.Med.Chem.(1992), 2798-2805; Berry, Nicola M. Synthesis (1986), 6, 476-480; Zenyuk, A. A. Chem.Nat.Compd.(Engl.Transl.) (1991), 27, 400-403; Mukheijee, Indian J.Chem.Sect.B (1984), 23, 193-198; Zenyuk, A. A., J.Org.Chem.USSR (Engl.Transl.) (1990), 26, 1926-1927; Demir, Tetrahedron Lett, (1989), 30, 1705-1708; Hamer, Tetrahedron Lett. (1986), 27, 2167-2168; Demir, J.Prakt.Chem./Chem.-Ztg. (1997), 339, 553-563; Rothstein, J.Chem.Soc. (1926), 2017; Landesberg, J.Org.Chem.(1968), 33, 3374-3382; Nilsson, Acta Chem.Scand.Ser.B (1981), 35, 667-668). 
Dihydropyridines of general formula (52), wherein Z, R1, R4, R5, R6, R7 and m are as defined in formula (I) and X is selected from Br or I, can be prepared as described in Scheme 11. Dicarbonyl compounds of general formula (10) can be treated with ammonia, followed by addition of (11) and ketosulfone (51) with heating in a solvent such as ethanol to provide dihydropyridines of general formula (52). An additional heating step, with an acid such as HCl, may be required to drive the reaction to completion. 
Ketosulfones of general formula (51), wherein R6, R7 and m are as defined in formula (I), can be prepared as described in Scheme 12. Ketones of general formula (54) can be treated with sodium borohydride (or the like) in a solvent such as ethanol to provide alcohols of general formula (55) which can be oxidized to the corresponding sulfone (56) using an oxidizing agent such as hydrogen peroxide catalyzed by sodium tungstate. Sulfones of general formula (56) can be treated with Jones"" reagent or the like to provide ketosulfones of general formula (51). 
Ketosulfones of general formula (66), wherein R6 and R7 are as defined in formula (I), can be prepared as described in Scheme 13. Thiols of general formula (58) wherein R is H or lower alkyl, can be reacted with an alkyl halide of general formula (59), wherein Rxe2x80x2 is H or lower alkyl to provide a sulfide of general formula (60), wherein R is H or lower alkyl, as described in (Fredga, Ark.Kemi (1943), 16 B, 8, 5; Challenger, J.Chem.Soc. (1959), 61, 68; Schoeberl, Justus Liebigs Ann. Chem.(1956), 599, 140, 151; Acheson, J.Chem.Soc.(1961), 650-660; Ghosh, J.Med.Chem. (1994), 37, 1177-1188). Sulfides of general formula (60) are also available via reaction between thiols of general formula (58) and unsaturated acids or esters of general formula (61) as described in (Reinhoudt, Synthesis, (1978) 368-370; Schoeberl, Justus Liebigs Ann. Chem.(1956), 599, 140, 151; Martani, Ric.Sci.(1959) 29, 520, 523; Dowd, Tetrahedron (1991), 47, 4847-4860; Fiesselmann, Chem.Ber.(1954), 87, 848, 854; Ranu, Tetrahedron (1992), 48, 1327-1332; Binder, Arch.Pharm.(Weinheim Ger.)(1981), 314, 557-564). A third method of generating sulfides of general formula (60) is via reaction of a thiol of general formula (63) wherein Rxe2x80x2 is H or lower alkyl, with an alkylhalide of general formula (62), wherein R is H or lower alkyl, as described in (Larsson, Chem.Ber. (1934), 67, 759; Kato, Chem.Pharm.Bull.(1986), 34, 486-495).
Sulfides of general formula (60) can be cyclized to provide sulfides of general formula (64) in the presence of a Lewis acid such as titanmium tetrachloride or a base such as sodium metal or an alkoxide. Examples of this transformation can be found in (Duus, Tetrahedron (1981), 37, 2633-2640; Deshmukh, Synth.Commun.(1996), 26, 1657-1662; Avison, Nature(London) (1944), 154, 459).
Sulfides of general formula (64) can be decarboxylated to provide sulfides of general formula (65) preferably in the presence of aqueous acid. Examples of this transformation can be found in (Ghosh, J.Med.Chem.(1994), 37, 1177-1188; Larsson, Sven.Kem.Tidskr.(1945), 57, 248; Woodward, J.Amer.Chem.Soc.(1946), 68, 2229, 2234). Sulfides of general formula (65) can be processed as described in Scheme 12 to provide ketosulfones of general formula (66). 
Enaminones of general formula (73), wherein Z, R1 and R2 are as defined in formula (I), can be prepared as described in Scheme 14. Dicarbonyl compounds (10) can be treated with an alcohol such as ethyl alcohol in the presence of an acid catalyst such as para-toluenesulfonic acid to provide vinyl ethers of general formula (70), wherein R is lower alkyl such as ethyl. Vinyl ethers of general formula (70) may contain a mixture of isomers which can be separated by a separatory method such as chromatography. Vinyl ethers of general formula (13) can be treated with ammonia in a solvent such as methanol to provide enaminones of general formula (73). 
Dihydropyridines of general formula (1), wherein A, D, Z, R1, R4, R5, R6, R7, m and n are as defined in formula (I) and X is Br or I, can be prepared as described in Scheme 15. Enaminones of general formula (73) can be treated with aldehydes of general formula (11) and carbonyl compounds of general formula (12) with heating in a solvent such as ethanol to provide dihydropyridines of general formula (1). An additional heating step, with an acid such as HCl, may be required to drive the reaction to completion. 
Dihydropyridines of general formula (49), wherein Z, R1, R4, R5, R6, R7 and m are as defined in formula (I) and X is selected from Br or I, can be prepared as described in Scheme 16. Enaminones of general formula (73) can be treated with aldehydes (11) and dicarbonyl compounds of general formula (48) with heating in a solvent such as ethanol in the presence of a base such as triethylamine to provide hemiaminals of general formula (83), dihydropyridines of general formula (49), or mixtures thereof. Hemiaminals (83) and mixtures containing hemiaminals (83) and dihydropyridines (49) may be treated with heat in the presence of an acid such as HCl in a solvent such as ethanol to effect complete conversion to dihydropyridines of general formula (49). 
An alternate method of preparing dihydropyridines of general formula (52), wherein Z, R1, R4, R5, R6, R7 and m are as defined in formula (I) and X is selected from Br or I, can be used as described in Scheme 17. Enaminones of general formula (73) can be treated with aldehydes of general formula (11) and ketosulfones of general formula (51) with heating in a solvent such as ethanol in the presence of a base such as triethylamine to provide hemiaminals of general formula (85), dihydropyridines of general formula (52), or mixtures thereof. Hemiaminals (85) and mixtures containing hemiaminals (85) and dihydropyridines (52) may be treated with heat in the presence of an acid such as HCl in a solvent such as ethanol to effect complete conversion to dihydropyridines of general formula (52). 
Dihydropyridines of general formula (90), wherein Z, R1, R4, R5, R6 and R7 are as defined in formula (I) and X is Br or I, can be prepared as described in Scheme 18. Enaminones of general formula (73) can be treated with aldehydes of general formula (11) and xcex2-ketoesters of general formula (87), wherein R is lower alkyl, to provide dihydropyridines of general formula (88). Dihydropyridines of general formula (88) can be treated with brominating agents such as N-bromosuccinimide or pyridinium tribromide in a solvent such as methanol, ethanol, isopropanol, or chloroform to provide dihydropyridines of general formula (89). Dihydropyridines of general formula (89) can be heated neat or heated in a solvent such as chloroform to provide dihydropyridines of general formula (90). 
An alternate method of preparing dihydropyridines of general formula (I), wherein A, D, Z, R1, R4, R5, R6, R7, m and n are as defined in formula (I) and X is Br or I, is described in Scheme 19. Dicarbonyl compounds of general formula (10) can be treated with aldehydes of general formula (11) and enamines of general formula (92) with heating in a solvent such as ethanol to provide dihydropyridines of general formula (1). An additional beating step, with an acid such as HCl, may be required to drive the reaction to completion. 
An alternate method of preparing dihydropyridines of general formula (49), wherein Z, R1, R4, R5, R6, R7 and m are as defined in formula (I) and X is Br or I, is described in Scheme 20. Dicarbonyl compounds of general formula (10) can be treated with aldehydes of general formula (11) and aminocycloalkenones of general formula (94) with heating in a solvent such as ethanol to provide dihydropyridines of general formula (49). An additional heating step, with an acid such as HCl, may be required to drive the reaction to completion. Aminocycloalkenones, such as 3-amino-2-cyclohexene-1-one, of general formula (94) can be purchased commercially (Fluka Chemical, Milwaukee, Wis.) or prepared as described in Kikani, B. Synthesis, (1991), 2, 176. Aminocycloalkenones of general formula (94) may also be prepared by treating dicarbonyl compounds of general formula (48) with ammonia in a solvent such as methanol. 
An alternate method of preparing dihydropyridines of general formula (90), wherein Z, R1, R4, R5, R6, and R7 are as defined in formula (I) and X is Br or I, is described in Scheme 21. Diones of general formula (10) can be treated with aldehydes of general formula (11) and compounds of general formula (96), wherein R is lower alkyl, to provide dihydropyridines of general formula (88) which can be processed as described in Scheme 18 to provide dihydropyridines of general formula (90). Compounds of general formula (96) can be generated from the reaction of xcex2-ketoesters of general formula (87) as described in Scheme 18 with ammonia. 
Enantiomers of general formula (94) and (95), wherein A, D, Z, R1, R4, R5, R6, R7, m and n are as defined in formula (I) and X is Br or I, may be prepared as single enantiomers by the method illustrated in Scheme 22. Dihydropyridines of general formula (1) can be treated with a base such as potassium tert-butoxide and (xe2x88x92)-8-phenylmenthol chloroformate in a solvent such as tetrahydrofuran to provide a mixture of diastereomeric carbamates (92) and (93). Diastereomers (92) and (93) can be separated by separatory methods known to those skilled in the art such as column chromatography over silica gel. The individual carbamates (92) and (93) following separation can be treated with sodium methoxide in methanol to produce the single enantiomers (94) and (95) respectively.
In addition to the methods illustrated in Scheme 22, racemic compounds of general formula (1) may be separated into individual enantiomers by chiral chromatography. Also, enantiomerically pure intermediates may be carried through Schemes 1-21 to provide enantiomerically pure [125I]dihydropyridines of general formula (3). For example, individual enantiomers may be synthesized from chiral diones of general formula (10).
Many of the starting benzaldehydes necessary to carry out the methods described in the preceeding and following Schemes may be purchased from commercial sources or may be synthesized by known procedures found in the chemical literature. Appropriate literature references for the preparation of benzaldehydes may be found in the following section or in the Examples. For starting materials not previously described in the literature the following Schemes are intended to illustrate their preparation through a general method.
The preparation of benzaldehydes used to synthesize compounds of the invention may be found in the following literature references: Pearson, Org. Synth. Coll. Vol V (1973), 117; Badder, J. Indian Chem. Soc. (1976), 53, 1053; Hodgson, J. Chem. Soc. (1927), 2425. 
Aldehydes of general formula (11), wherein R1 is as defined in formula (I) and X is selected from Br or I, can be prepared according to the method described in Scheme 23. A para substituted compound of general formula (100) wherein R is COOH, CHO, CH2OH or COORxe2x80x2, wherein Rxe2x80x2 is lower alkyl, may by nitrated to provide compounds of general formula (101). Nitration on compounds of general formula (100) are well known to those skilled in the art. Nitrated compounds of general formula (101) can be reduced to the corresponding anilines of general formula (102) using methods known to those skilled in the art. Anilines of general formula (102) can be converted to bromides and iodides of general formula (103) using the Sandmeyer reaction. The Sandmeyer reaction involves converting anilines of general formula (102) to an intermediate diazonium salt with sodium nitrite. The diazonium salts can be treated with an appropriate source of bromine or iodine to provide the bromide or iodide. The Sandmeyer reaction and conditions for effecting the transformation are well known to those skilled in the art of organic chemistry. Compounds of general formula (103) can be converted to aldehydes of general formula (11) using reductive (reduce ester or acid) or oxidative (oxidize alcohol) procedures well known to those skilled in the art. 
Aldehydes of general formula (11), wherein R1 is as defined in formula (I) and X is selected from Br or I, can also be prepared according to the method described in Scheme 24. A para substituted compound of general formula (100) wherein R is COOH, CHO, CH2OH or COORxe2x80x2, wherein Rxe2x80x2 is lower alkyl, may by subjected to conditions of an electrophilic aromatic substitution reaction to provide bromides or iodides of general formula (103). Compounds of general formula (103) can be converted to aldehydes of general formula (11) using reductive (reduce ester or acid) or oxidative (oxidize alcohol) reductive (reduce ester or acid) or oxidative (oxidize alcohol)procedures well known to those skilled in the art.